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Selected Protocol from "Protein-Protein Interactions", 2nd Edition, E. Golemis, P. Adam.
Phosphorylation of Substrates
Scott T. Eblin, N. Vinay Kumar and Michael J. Weber
Department of Microbiology and Cancer Center, Universtiy of Virginia Health System, Charlottesville, VA 22908
ABSTRACT
Ideally, one would like to be able to directly phosphorylate substrates in an intact cell. This could potentially be performed by introducing ATP analog into live or permeabilized cells. However, cells are impermeable to ATP, and the addition of labeled ATP analog to digitonin-permeabilized cells results in the hydrolysis of the analog ATP within 1 min (Chaudhary et al. 2002). Therefore, we have chosen to apply this technique to cell lysates or fractions. Here we describe two approaches to detect direct substrates of a protein kinase. The first approach involves identification of kinase-associated substrates by immunoprecipitating a tagged form of the mutant kinase from transfected COS-1 cells and performing a kinase reaction by the addition of [g-32P]ATP analog. The second approach involves the addition of recombinant mutant kinase and [g-32P]ATP analog to cell lysates. We have used these techniques for the phosphorylation of ERK2 substrates; however, this methodology can be applied to other protein kinases |
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Products Available for this Protocol
| Protocol Material Description |
Product # |
Product Name |
Add to Cart |
| Recombinant kinase |
C8482 |
Protein Kinase A catalytic subunit human, >90% (SDS-PAGE), recombinant, expressed in
Escherichia coli, buffered aqueous glycerol solution |
|
| Sepharose CL-6B beads |
CL6B200 |
Sepharose® CL-6B, Cross-linked |
|
| SDS |
L4390 |
Sodium dodecyl sulfate, for molecular biology |
|
| PBS |
P5368 |
Phosphate buffered saline, pH 7.4, Biotechnology Performance Certified |
|
| Serum-free medium (DMEM) |
D6421 |
Dulbecco’s Modified Eagle’s Medium/Nutrient Mixture F-12 Ham, With 15 mM HEPES and sodium
bicarbonate, without L-glutamine, liquid, sterile-filtered, cell culture tested |
|
| FLAG Peptide |
F3290 |
FLAG® Peptide, lyophilized powder |
|
| NaCl |
S3014 |
Sodium chloride, for molecular biology |
|
| 2x Laemmli sample buffer |
S3401 |
Sample Buffer, Laemmli 2× Concentrate |
|
| 20 mM HEPES (pH 7.4) |
51558 |
HEPES buffered saline, BioChemika Ultra, for molecular biology, 2× concentrate
|
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| 2mM EGTA |
E3889 |
Ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid, for molecular
biology |
|
| 2mM MgCl2 |
M4880 |
Magnesium chloride |
|
| COS-1 cells |
88031701 |
COS-1 Cell Line from African green monkey kidney, SV40 transformed |
|
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